Radiolabled cyclopamine assay for the smoothened receptor

Abstract

The present invention discloses novel methods for screening compositions for both agonist and antagonist activity on the smoothened receptor. The assay tests ligands on the activity of the receptor using a cyclopamine binding and ultra high receptor expression.

1 . A method for determining if a test compound is a ligand of the Smoothened receptor comprising combining the test compound with a radiolabeled cyclopamine or a derivative thereof and exposing them to one or more cells expressing over expressing the Smoothened receptor at a rate of at least 200,000 receptors per cell and determining the competition binding of the test compound. 
     
     
         2 . A method for assessing the binding of a test composition to the Smoothened receptor comprising:
 a) selecting a radiolabeled cyclopamine ligand or a cyclopamine derivative ligand thereof;   b) preparing a plurality of cells that over express the Smoothened receptor at a rate of at least 200,000 receptors per cell;   c) determining the binding of the selected radiolabeled ligand to the cells without the presence of a test compound;   d) exposing a selected concentration of radiolabeled ligand and test compound to a selected amount of the cells over expressing the Smoothened receptor; and   e) comparing the amount of radiolabeled ligand binding to the cells without the presence of the test compound with the amount of radiolabeled ligand binding to cells in the presence of the test compound to determine the binding ability of the test compound.